This project will examine the effect of circulating iron produced from transfusions of older, stored red blood cells (RBCs) on biofilm-related infections. By FDA criteria, RBCs have a maximum refrigerated shelf life of 42 days. Studies suggest that transfusion of older versus fresher stored RBC units is associated with significantly increased risk of morbidity and mortality. RBCs are damaged progressively during refrigerator storage. After transfusion, storage-damaged RBCs are rapidly cleared from the circulation by macrophages. This RBC hemoglobin iron is then rapidly catabolized and returned to plasma at a pace that can exceed the rate of iron uptake by transferrin, the physiologic iron transporter, and thereby producing circulating non-transferrin- bound iron. Physiologically, virtually all circulating iron is transferrin-bound. In contrast, our human studies show that transfusion of even one unit of stored RBCs can acutely produce circulating non-transferrin-bound iron and enhance bacterial growth in post-transfusion serum samples in vitro. Because iron transport by transferrin is a critical host defense strategy that withholds iron from infectious pathogens, our innovative hypothesis is that release of iron after acute macrophage clearance of storage-damaged RBCs overwhelms the recipient's capacity for safe iron sequestration, thereby producing circulating non- transferrin-bound iron that then acts as a virulence factor by enhancing biofilm formation. Biofilms are aggregates of microorganisms growing in a community embedded in an extracellular polymeric substance. In many microbial systems, differentiation into a biofilm requires high levels of iron. Biofilm-related infections are responsible for importat healthcare-associated infections, such as central line-associated blood stream infections (CLABSI). This proposal will determine the extent to which circulating iron produced by clearance of transfused RBCs enhances biofilm formation of microbial pathogens. Aim #1 will use all pediatric and adult incident cases of CLABSI in our hospital system to conduct an innovative case-crossover and case- time-control retrospective study to determine the extent of the association between the duration of RBC storage prior to transfusion and CLABSI. In Aim #2, we will use static and dynamic in vitro biofilm assays to determine the contribution of non-transferrin-bound iron to biofilm formation of several relevant human pathogens. The current project will fill critical gaps in knowledge by providing evidence for a relationship between transfusions of stored RBCs and CLABSI, and determining the contribution of iron to the underlying mechanism(s) responsible for this effect. This will lead to innovative approaches for preventing production of circulating iron, thereby improving RBC transfusion safety in hospitalized patients.